PROJECT SUMMARY/ABSTRACT This application is for an upgrade to our current Bruker Q-band E580 pulse spectrometer capable of running DEER (double electron electron resonance, or pELDOR), DQC (double quantum coherence), and ENDOR (electron nuclear double resonance) experiments at cryogenic temperatures. New state-of-the-art components (300 W TWT amplifier, SpinJet AWG, and E5106QT-II resonator) are requested to enable the detection of lower concentrations of sample and longer distances, enable the use of copper as a spin probe, enhance the quality of the raw data and dramatically decrease the background signals for more accurate analysis and results, and allow ELDOR-detected NMR studies. The primary use of both the current and upgraded instrument is to quantitate distance measurements between paramagnetic probes on or within biomedically relevant proteins and peptides. The increased concentration sensitivity, signal-to-noise ratios, and modulation depths will be the result of shorter, shaped pulses flipping more of the spin population; the ability to routinely run at <80K; shorter acquisition times; and improvement in resolution, accuracy, identification, and collection of longer distances. Each of the upgraded components, together with the institutional purchase of a cryogen-free system, will provide a significant improvement in data collection as well as a tremendous advantage (>10-fold increase in sensitivity) over our current technology. These advantages will benefit an array of largely NIH-funded biological projects and enable studies on protein systems and sample concentrations not attainable with our current instrumentation. The combination of the requested components will provide a remarkable advantage to biological research projects and, coupled with the considerable improvement in data accuracy, will prove to have a substantial impact on current and future structural biology studies of soluble and membrane proteins and protein complexes. This type of state-of-the-art Q-band pulsed EPR instrumentation is lacking not only at the Medical College of Wisconsin but in the entire the state of Wisconsin. Six major and nine minor user groups are showcased who will measure long-range distances within proteins and protein complexes as well as carry out ELDOR-detected NMR, all with a need for the requested state-of-the-art advanced pulse instrumentation not available in the region. Specifically, the research proposed here, using these novel commercially available enhancements to a biophysical spectroscopic technique, will enable the study of protein structure and functional dynamics that will lead to a better understanding of the physiology of disease processes such as cardiovascular and pulmonary diseases; cystic fibrosis; diabetes; obesity; behavioral, neurological, and psychiatric disorders; Alzheimer?s disease; and cancer. It will also contribute to the development of novel antibiotics and cancer therapeutic agents, and to the design of safer and more effective drugs targeting a broad spectrum of diseases. This upgraded instrumentation capability will immediately and significantly advance the productivity of the NIH-funded and other projects outlined in this proposal as well as enhance the research environment for the surrounding community.